RESUMEN

During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in G2 phase by a ß-catenin-independent (Wnt/STOP) Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical ß-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine kinases EGFR and Abelson kinase (Abl), which promote nuclear entry of ß-catenin and facilitate exit from G2. This strategy combines transcription-dependent and -independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

RESUMEN

Objective @#This article explores the relationship between congenital tooth agenesis and related gene mutations, providing a reference for early diagnosis of the disease.@*Methods @# Clinical and radiographic examinations of a rare case of congenital tooth agenesis were conducted to evaluate the abnormal morphology and quantity of the teeth, as well as the overall health of the patient. Bidirectional sequencing of the PAX9 and MSX1 genes and whole-exome sequencing were conducted to identify potential genetic abnormalities. Sanger sequencing of the newly discovered mutation site was performed on the proband's son. Subsequently, the impacts of the mutations were evaluated through computational tools and a cell-based gene transfection assay. @*Results @#This is a rare case of tooth agenesis characterized by a congenitally missing first molar, a second molar with one single root and a supernumerary second premolar in the right mandibular dentition. The c.717 C>C/T in PAX9 is synonymous. The c.119C>G in MSX1 is a missense mutation predicted to be “benign” by Polyphen. Through whole-exome sequencing, we found a novel mutation, c.637-7 C>A in intron 3 of the WNT6 gene, which is predicted by MAXENT to influence the splicing of mRNA. Both the proband and his son carry this mutation. A cell-based gene transfection assay demonstrated that it did not alter the mRNA splicing of WNT6. @* Conclusion @#The interaction between single nucleotide polymorphisms may contribute to congenital tooth agenesis.

RESUMEN

Wnt/ß-catenin pathway is associated with the progression of various cancers such as gastric cancer, colorectal cancer, and endometrial cancer. Using the Kaplan-Meier Plotter database, we found that WNT6 was associated with progression-free survival (PFS) outcomes. Immunohistochemical analysis of ovarian cancer samples and normal ovaries showed that the expression of WNT6 protein was significantly increased in ovarian cancer samples. Further, we explored the possible role of WNT6 in the occurrence and development of ovarian cancer. Our results showed that the mRNA and protein expression of WNT6 were significantly higher in CAOV3 and OVCAR3 cells compared with other ovarian cancer cell lines and normal ovarian cell line IOSE-80 as well. The transformation of CAOV3 and OVCAR3 cells with short interfering WNT6 (siWNT6) significantly inhibited their proliferation and lamellipodia formation, causing cell cycle arrest and promoting cell apoptosis. Western blot experiments confirmed that the down-regulation of WNT6 inhibited the expression of ß-catenin and Notch1. These results suggest that WNT6 plays an important role in the occurrence and development of ovarian cancer.

Asunto(s)

Neoplasias Ováricas , Vía de Señalización Wnt , Apoptosis , Carcinoma Epitelial de Ovario/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/metabolismo , ARN Mensajero , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

Preeclampsia (PE), a systemic vascular disorder, is the leading cause of maternal and perinatal morbidity and mortality, and its pathogenesis has yet to be fully elucidated. Siglec6, a transmembrane protein, is highly expressed in human placental trophoblasts, and previous studies have shown that Siglec6 overexpression correlates with PE, but the role of Siglec6 during PE progression is unknown. Here, we demonstrated that the mRNA and protein expression levels of Siglec6 were upregulated in early-onset PE placentas compared with uncomplicated pregnancies, and Siglec6 was primarily located in syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). Moreover, our results showed that chemical reagent-induced HIF-1α accumulation promoted the mRNA and protein levels of Siglec6 in HTR8/SVneo and BeWo cells. Although Siglec6 overexpression did not affect HTR8/SVneo cell proliferation, migration, and invasion, the conditional medium derived from the Siglec6 overexpressed HTR8/SVneo cells (Siglec6-OE-CM) significantly impaired the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, the transcriptome sequencing results revealed that Siglec6 overexpression led to the downregulation of Wnt6 in HTR8/SVneo cells, which was further confirmed by qPCR and ELISA. Recombinant human Wnt6 reversed Siglec6-OE-CM-mediated suppression of HUVEC functions by reactivating the Wnt/ß-catenin signaling pathway. Altogether, our study found that elevated trophoblastic Siglec6 contributed to the impairment of vascular endothelial cell functions by downregulating Wnt6/ß-catenin signaling.

Asunto(s)

Antígenos de Diferenciación Mielomonocítica , Lectinas , Preeclampsia , Trofoblastos , Femenino , Humanos , Embarazo , beta Catenina/metabolismo , Línea Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , ARN Mensajero/metabolismo , Trofoblastos/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos CD , Lectinas/genética

RESUMEN

OBJECTIVE: The aim of this study was to test the hypothesis in vitro and in vivo, that the glycoprotein Wnt6 can regulate human dental papilla cell differentiation by ß-catenin signaling. DESIGN: The expression of Wnt6 was detected by quantitative polymerase chain reaction (qPCR). Wnt6 stealth RNA was used to knockdown the expression of Wnt6. The Wnt canonical signaling was detected by immunofluorescence staining, qPCR, and TOPflash/FOPflash dual-luciferase reporter assay. The differentiation was investigated by alkaline phosphatase staining or Alizarin Red staining after osteo/odontogenic medium culture and by Masson trichrome staining after subcutaneous transplantation. There are at least three samples in one group for each experiment. RESULTS: Wnt6 protein and mRNA were high expressed in dental mesenchyme tissue and cells. In human dental papilla cells, Wnt6 over-expression could activate ß-catenin dependent pathway, including ß-catenin accumulation in cell nuclei, lymphoid enhancer factor 1 mRNA up-regulation, and enhanced ß-catenin transcriptional activity. Wnt6 activated ß-catenin pathway in a similar way to Wnt3a but at a lower level. Wnt6 inhibited human dental papilla cells differentiation as alkaline phosphatase activity in vitro, and promoted differentiation as mineralization after subcutaneous transplantation in vivo, as same trend as Wnt3a but at a lower level. The Wnt/ß-catenin inhibitor XAV939 treatment attenuated Wnt6- or Wnt3a-induced human dental papilla cells mineralization. CONCLUSIONS: Wnt6 activated ß-catenin dependent pathway and regulated human dental papilla cells differentiation. Potential mechanism of Wnt6-regulated cell differentiation is the activation of Wnt/ß-catenin signaling pathway.

Asunto(s)

Vía de Señalización Wnt , beta Catenina , Fosfatasa Alcalina/metabolismo , Diferenciación Celular/fisiología , Papila Dental , Glicoproteínas , Humanos , Osteogénesis , ARN Mensajero , Proteínas Wnt , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo

RESUMEN

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFß1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFß1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFß1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFß1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFß1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Asunto(s)

Células Endoteliales , Factor 2 de Crecimiento de Fibroblastos , Factor de Crecimiento Transformador beta1/fisiología , Animales , Proteína Morfogenética Ósea 4 , Diferenciación Celular , Células Madre Embrionarias , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratones , Proteínas Proto-Oncogénicas , Células del Estroma , Factor de Crecimiento Transformador beta1/genética , Proteínas Wnt

RESUMEN

The uterus, as part of the female reproductive tract, is essential for embryo survival and in the maintenance of multiple pregnancies in domestic animals. This study was conducted to investigate the effects of WNT6 on Hu sheep endometrial epithelial cells (EECs) and uterine glands (UGs) in Hu sheep, with high prolificacy rates. In the present study, Hu sheep with different fecundity, over three consecutive pregnancies, were divided into two groups: high prolificacy rate group (HP, litter size = 3) and low prolificacy rate group (LP, litter size = 1). A comparative analysis of the endometrial morphology was performed by immunofluorescence. RNA-seq was used to analyze the gene's expression in endometrium of HP and LP Hu sheep, providing a candidate gene, which was investigated in EECs and organoid culture. Firstly, higher density of UGs was found in the HP Hu sheep groups (p < 0.05). The RNA-seq data revealed the importance of the WNT signaling pathway and WNT6 gene in Hu sheep endometrium. Functionally, WNT6 could promote the cell cycle progression of EECs via WNT/ß-catenin signal and enhance UGs organogenesis. Taken together, WNT6 is a crucial regulator for sheep endometrial development; this finding may offer a new insight into understanding the regulatory mechanism of sheep prolificacy.

RESUMEN

The homeostasis of the stem cell niche is regulated by both intrinsic and extrinsic factors, and the complex and ordered molecular and cellular regulatory mechanisms need to be further explored. In Drosophila testis, germline stem cells (GSCs) rely on hub cells for self-renewal and physical attachment. GSCs are also in contact with somatic cyst stem cells (CySCs). Utilizing genetic manipulation in Drosophila, we investigated the role of Wnt6 in vivo and in vitro. In Drosophila testis, we found that Wnt6 is required for GSC differentiation and CySC self-renewal. In Schneider 2 (S2) cells, we found that Wnt6 regulates cell proliferation and apoptosis. Mechanistically, we demonstrated that Wnt6 can downregulate the expression levels of Arm, Rac1 and Cdc42 in S2 cells. Notably, Rac1 and Cdc42, which act downstream of the noncanonical Wnt signalling pathway, imitated the phenotypes of Wnt6 in Drosophila testis. Thus, the newly discovered Wnt6-Rac1/Cdc42 signal axis is required for the homeostasis of the stem cell niche in the Drosophila testis.

Asunto(s)

Proteínas de Drosophila/genética , Proteínas de Unión al GTP/genética , Testículo/crecimiento & desarrollo , Proteínas Wnt/genética , Proteínas de Unión al GTP rac/genética , Animales , Apoptosis/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Células Germinativas/metabolismo , Homeostasis/genética , Masculino , Nicho de Células Madre/genética , Células Madre/metabolismo , Testículo/metabolismo

RESUMEN

Obesity causes a wide range of metabolic diseases including diabetes, cardiovascular disease, and kidney disease. Thus, plenty of studies have attempted to discover naturally derived compounds displaying anti-obesity effects. In this study, we evaluated the inhibitory effects of morolic acid 3-O-caffeate (MAOC), extracted from Betula schmidtii, on adipogenesis. Treatment of 3T3-L1 cells with MAOC during adipogenesis significantly reduced lipid accumulation and decreased the expression of adiponectin, a marker of mature adipocytes. Moreover, the treatment with MAOC only during the early phase (day 0-2) sufficiently inhibited adipogenesis, comparable with the inhibitory effects observed following MAOC treatment during the whole processes of adipogenesis. In the early phase of adipogenesis, the expression level of Wnt6, which inhibits adipogenesis, increased by MAOC treatment in 3T3-L1 cells. To identify the gene regulatory mechanism, we assessed alterations in histone modifications upon MAOC treatment. Both global and local levels on the Wnt6 promoter region of histone H3 lysine 4 trimethylation, an active transcriptional histone marker, increased markedly by MAOC treatment in 3T3-L1 cells. Our findings identified an epigenetic event associated with inhibition of adipocyte generation by MAOC, suggesting its potential as an efficient therapeutic compound to cure obesity and metabolic diseases.

Asunto(s)

Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Epigénesis Genética/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Triterpenos/química , Triterpenos/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ratones , Estructura Molecular , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genética

RESUMEN

Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) gene, the gene encoding eIF2γ protein, is globally expressed in all tissues and plays important roles in regulating global and gene-specific mRNA translation initiation. It has been noticed that Eif2s3y plays crucial roles in spermatogenesis, however, the mechanism remains unclear. In the current study, transgenic Eif2s3y mice were generated to test our hypothesis that the Eif2s3y promotes the proliferation of spermatogonial stem cells (SSCs). Transgenic Eif2s3y mouse had enhanced SSCs proliferation rate when compared to WT mouse. Interesting, the testes from transgenic Eif2s3y mouse had increased Active-ß-catenin protein expression and higher expression pattern of Wnt ligand Wnt6 when compared to testes from WT mouse. This study revealed novel roles of Eif2s3y in the activation Wnt6/ß-catenin signal pathway in SSCs. Taken together, we identified Eif2s3y-Wnt6-ß-catenin as a critical pathway in the regulation of spermatogenesis, which provides a platform for investigating the molecular mechanisms of male reproduction.

Asunto(s)

Proteínas Proto-Oncogénicas/metabolismo , Espermatogonias/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Línea Celular , Proliferación Celular , Masculino , Ratones Transgénicos , Modelos Biológicos

RESUMEN

Wnt genes encode secreted ligands that play many important roles in the development of metazoans. There are thirteen known Wnt gene subfamilies and seven of these are represented in Drosophila melanogaster. While wingless (wg) is the best understood and most widely studied Wnt gene in Drosophila, the functions of many of the other Drosophila Wnt genes are less well understood. For example, relatively little is known about Wnt6, which is an ancient paralog of wg and they form a conserved Wnt cluster together with Wnt9 (Dwnt4) and Wnt10. Wg and Wnt6 encode similar proteins and exhibit overlapping expression in several tissues during development. Both wg and Wnt6 were previously shown to regulate the development of maxillary palps, important olfactory organs in flies, but it remained unclear how these two ligands may combine to carry out specific functions and how this is regulated. Here, we have further analysed Wnt6 function in the context of maxillary palp development. Surprisingly, we found that Wnt6 does not appear to be necessary for development of maxillary palps. While a deletion of the 5' region of Wnt6 results in very small maxillary palps, we show that this effect is more likely to be a consequence of removing cis-regulatory elements that may regulate wg expression in this tissue rather than through the loss of Wnt6 function. Although, we cannot completely exclude the possibility that Wnt6 may subtly regulate maxillary palp development in combination with wg, our analysis of Wnt6 loss of function mutants suggests this ligand plays a more general role in regulating growth during development. Taken together our results provide new insights into maxillary palp formation and Wnt6 functions in Drosophila, and further evidence for a complex cis-regulatory landscape in the Wnt9-wg-Wnt6-Wnt10 cluster, which may help explain its evolutionary conservation.

Asunto(s)

Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Genes de Insecto/genética , Vías Olfatorias/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transducción de Señal/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología

RESUMEN

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

Asunto(s)

Centrosoma/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteínas 14-3-3/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Glucemia/metabolismo , Centrosoma/patología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Exorribonucleasas/metabolismo , Femenino , Receptores Frizzled/genética , Productos Finales de Glicación Avanzada/farmacología , Células HCT116 , Humanos , Insulina/sangre , Ratones Endogámicos ICR , Ácido Palmítico/farmacología , Unión Proteica , Ratas , Proteínas Wnt/genética , Quinasas Asociadas a rho/metabolismo

RESUMEN

WNT family member 6 (WNT6) is a member of the highly conserved WNT protein family. It plays an essential role in the normal development process, not only in embryonic morphogenesis, but also in post-natal homeostasis. WNT6 functions in mice and humans. This review summarizes the current findings on the biological functions of WNT6, describing its involvement in regulating embryogenesis, decidualization, and organ development. Aberrant WNT6 signaling is related to various pathologies, such as promoting cancer development, lung tuberculosis, and kidney fibrosis and improving the symptoms of Rett syndrome (RTT). Thus, due to its various functions, WNT6 has great potential for in-depth research. This work not only describes the signaling mechanism and function of WNT6 under physiological and pathological conditions, but also provides a theoretical basis for targeted therapy.

RESUMEN

Objective: As a member of the Wnt family, WNT6 contributes to tumorigenesis and the development of various types of cancer. However, the expression status of WNT6 in colorectal liver metastasis (CRLM) and its prognostic value remain to be elucidated. In this study, we evaluated the association of WNT6 expression with survival outcomes in CRLM patients undergoing liver resection. Methods: The medical records of 106 consecutive CRLM patients undergoing curative tumor resection between October 1996 and December 2011 were retrospectively selected. WNT6 expression was detected using immunohistochemistry (IHC) analyses on paraffin-embedded specimens. The IHC score was determined according to the percentage and intensity of positively stained cells. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and the log-rank test, and independent prognostic factors were determined by Cox regression modeling. Results: We found that WNT6 was commonly expressed in 93.4% (99/106) of colorectal cancer tissues. The median IHC score of WNT6 expression was significantly lower in patients receiving preoperative chemotherapy than those without preoperative chemotherapy (1.33 vs. 2.33, P = 0.033). Survival analysis indicated that patients with high WNT6 expression had poorer 5-year OS than those with low WNT6 expression (31.0% vs. 62.2%, P = 0.012). The 5-year OS rate was significantly lower in the high WNT6 group than in the low WNT6 group (36.8% vs. 79.9%, P = 0.013) in low-risk patients but was comparable among the high-risk patients (22.7% vs. 34.7%, P = 0.433). Multivariate analysis indicated that high WNT6 expression was independently associated with poor OS (hazard ratio [HR]: 2.089; 95% confidence interval [CI]: 1.231-3.545; P = 0.006). Conclusions: High expression of WNT6 was associated with unfavorable oncologic prognosis in patients with CRLM undergoing liver resection. Detection of WNT6 expression may be valuable for guiding postoperative treatment.

RESUMEN

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo. Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor.

Asunto(s)

Biomarcadores/análisis , Glioblastoma/diagnóstico , Glioblastoma/patología , Proteínas Wnt/análisis , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Pronóstico , Proteínas Proto-Oncogénicas/análisis , Transducción de Señal , Análisis de Supervivencia , Temozolomida/farmacología , Proteínas Wnt/genética

RESUMEN

Wnt proteins have been reported to contribute to the progression of various types of cancer. Wnt6 is a member of the Wnt family and may promote tumorigenesis in gastrointestinal cancer and cervical cancer. In the present study, the expression of Wnt6 in human colon cancer cell lines was evaluated, in order to investigate the role of Wnt6 in the development of colon cancer. Additionally, the effects of Wnt6 upregulation or downregulation on proliferation, apoptosis, cell cycle and cell migration of colon cancer cells have been investigated. Furthermore, western blot analysis was employed to evaluate the expression of Wnt6, B-cell lymphoma 2-associated X protein (Bax), caspase-3 and matrix metalloproteinase (MMP)2. The results of the present study demonstrated that the expression of Wnt6 was increased in HCT116 and SW480 cells compared with the remaining colon cancer cell lines. Furthermore, overexpression Wnt6 resulting from transfection of pGPU6/GFP/Neo-Wnt6-Homo-1 plasmid promoted the proliferation, cell cycle and migration of HCT116 and SW480 cells, but inhibited cell apoptosis in vitro. The expression of caspase-3 and MMP2 was increased, whereas the expression of Bax was decreased in response to upregulation of Wnt6. These results suggested that Wnt6 may serve a vital function in the development of colon cancer.

RESUMEN

The aim of this study was to evaluate the status and explore the impact of DNA methylation of WNT6 gene with osteosarcoma in children. A total of 50 patients with primary osteosarcoma in children were enrolled. The expression of WNT6 protein was determined by immunohistochemical staining. The DNA methylation level of WNT6 gene was evaluated by methylation-specific PCR (MSP). Human MG63 osteosarcoma cells and human normal Hfob1.19 osteoblasts were selected and cultured. Western blot analysis was utilized to measure the expression of WNT6 in the two cell lines. MSP was used to assess the status of DNA methylation of WNT6 gene. The correlation between DNA methylation of WNT6 gene and prognosis of osteosarcoma in children was evaluated by statistical analysis of the clinical and prognostic data. The results of the immunohistochemical assay showed that 84% (42/50) of primary osteosarcoma was WNT6 positive. MSP indicated that DNA methylation of WNT6 gene was found in 9 cases (18%) of primary osteosarcoma. Western blot analysis showed that WNT6 protein expression in MG63 cells was significantly higher than that in normal human Hfob1.19 osteoblasts, whereas, the level of DNA methylation of WNT6 gene in MG63 cells was significantly lower than in Hfob1.19 cells. Analysis of survival, prognosis and their correlation found that the DNA methylation level of WNT6 gene was negatively correlated with the prognosis of children with osteosarcoma. In conclusion, there was a high level of WNT6 gene expression in primary osteosarcoma, which was mainly due to low DNA methylation level of WNT6 gene. The DNA methylation of WNT6 gene was negatively correlated with the prognosis of patients with osteosarcoma in children.

RESUMEN

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3ß (Ser9), nuclear accumulation of ß-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-ß (TGF-ß); Wnt6 reversed TGF-ß-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-ß-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/ß(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.

Asunto(s)

Diferenciación Celular/genética , Enfermedades Renales/genética , Enfermedades Renales/patología , Riñón/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/genética , Proteínas Wnt/fisiología , Animales , Células Epiteliales/patología , Femenino , Fibrosis , Receptores Frizzled , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas I-kappa B/genética , Riñón/embriología , Enfermedades Renales/inducido químicamente , Túbulos Renales/crecimiento & desarrollo , Ratones , Ratones Noqueados , Fosforilación , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética , Factor de Transcripción ReIA/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Vimentina/biosíntesis

RESUMEN

WNT proteins are a family of secreted, cysteine-rich proteins containing 19 members. Signaling through WNT proteins is reported to be involved in carcinogenesis and cancer progression of gastrointestinal tumors, such as gastric cancer and colon cancer. The expression status of WNT6 in ESCCs and their clinico-prognostic significances remain to be elucidated. In this study, One-hundred and thirty-six patients with ESCC were explored. Paraffin-embedded tumor sections were stained with WNT6 antibody. The correlations between WNT6 expression and survival parameters were analyzed. The overall frequency of WNT6 over-expression was 50.7% (69/136) of advanced EC patients. For DMS and OS, over-expression of WNT6 remained the independent factor for worse prognosis (hazard ratio (HR), 2.425; 95% CI, 1.631-3.605; P < 0.001 for OS and HR, 2.238; 95% CI, 1.507-3.323; P < 0.001 for DMS, respectively). To conclude, our results support the concept that WNT6 may play a role in tumor progression. WNT6 over-expression inversely correlates with the poor long-term survival in ESCC patients. WNT6 can be considered as a predictor for recurrence.

Asunto(s)

Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Proteínas Wnt/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/mortalidad , Progresión de la Enfermedad , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Wnt/análisis

RESUMEN

STUDY QUESTION: Do mutations and/or polymorphisms in coding sequences in Wingless-Type MMTV Integration Site Family, Member 6 (WNT6) play a role in unexplained recurrent miscarriage (unexplained RM) in Chinese couples? SUMMARY ANSWER: We found four mutations in the coding sequences of WNT6 which appear to exist in a small proportion of Chinese women with unexplained RM. WHAT IS KNOWN ALREADY: WNT6 has been proved to be essential for stromal cell proliferation during decidualization in mice, but in humans WNT6 has not been studied in recurrent miscarriage populations. STUDY DESIGN, SIZE, DURATION: For this study, 100 couples with unexplained RM (at least three or more unexplained spontaneous miscarriages), and 100 ethnically matched fertile couples (at least one live birth and no history of pregnancy pathologies) were recruited. All the participants were chosen over a 7-year period from the National Research Center for Assisted Reproductive Technology and Reproductive Genetics at Shandong University, Jinan, China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were recruited following extensive clinical studies. Genomic DNA was isolated from peripheral blood. Mutation analysis in the coding regions of WNT6 was performed by PCR amplification and DNA sequences testing in all participants. Functional effects of missense variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT). MAIN RESULTS AND THE ROLE OF CHANCE: Four rare novel mutations, including one missense mutation, were found in intron 1, exon 3 and the 3' untranslated region of WNT6 in four women with unexplained RM. Gene software predictions showed that the missense mutation in exon 3 could alter the function of WNT6. No mutations or polymorphisms were detected in the male partners of the unexplained RM patients or in the fertile controls. To further validate the findings, we continued to screen this missense mutation site in another 100 peripheral blood samples of normal fertile females, and there was still no positive result. LIMITATIONS, REASONS FOR CAUTION: There is no direct evidence to validate whether these novel mutations discovered in the present research are related to unexplained RM. Further studies are warranted to investigate the role of WNT6 in unexplained RM, including larger studies in an independent group. WIDER IMPLICATIONS OF THE FINDINGS: These results provide evidence to suggest the importance of WNT6 in reproductive failure and may support the hypothesis that WNT6 is essential for stromal cell proliferation during decidualization. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Science and Technology Development Planning of Shandong (2013GGE27001), the National Natural Science Foundation of China (81300459), the Science Projection of Bureau of Public Health in Weifang (2012044) and the Science Research Foundation Item of No-earnings Health Vocation. The authors have no competing interests to declare.

Asunto(s)

Aborto Habitual/genética , Mutación Missense , Proteínas Wnt/genética , Regiones no Traducidas 3' , Secuencia de Bases , Proliferación Celular , China , Exones , Femenino , Fertilidad , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Mutación , Polimorfismo Genético , Análisis de Secuencia de ADN